Field of the Invention
The present invention is related to a reversible, parallel and/or multitask cloning method and kit, which improves the cloning of (preferably multiple) genetic element(s) in a nucleic acid construct such as a vector or the chromosome of a cell and the rapid and efficient selection of constructs with a correct integration of said genetic element(s), either in vitro or in vivo.
More precisely, the present invention is related to integrated method and tools to construct recombinant DNA molecules (to be used as DNA vaccine or for gene therapy) without requiring the use of antibiotic(s) resistance gene(s) and without requiring the addition of one or more antibiotic(s) to the culture medium of cells submitted to this recombinant DNA method. The present invention allows to obtain the selection of recombinant host cell(s) transformed by a (exogenous) nucleic acid sequence of interest (extra-chromosomal vector containing the insert) and simultaneously stabilization (stable inheritance) of this (exogenous) nucleic acid sequence of interest into the transformed host cell(s) descendants (maintenance of the nucleic acid sequence of interest in the host cells population).
To obtain complex molecular constructs comprised of multiple genetic elements, the selection of the genetic events (insertion(s) and/or deletion(s) and/or inversions(s) of DNA fragments) that will cause the assemblage of the target construct comprised of the said genetic elements at the right position and with the right orientation is usually a time consuming procedure.
In particular, one is necessary faced with the major problem of selecting different multiple genetic events (insertion, deletion, inversion of a genetic sequence in a nucleic acid construct), possibly in the same reaction tube.
Therefore, a molecular biologist should usually obtain a genetic event (insertion, deletion, inversion of a genetic sequence in a nucleic acid construct) separately and not simultaneously in the same reaction tube and should avoid any mistake (incorrect integration of a genetic sequence in the wrong direction, etc.) during said genetic manipulation.
Generally, DNA cloning is done in two steps: firstly, a DNA fragment is inserted in vitro in a vector (i.e. an autonomously replicating genetic construct, such as a virus or a plasmid) and thereafter, a mixture containing vectors with insert and vectors alone are introduced into bacteria to increase the number of DNA molecules, these molecules are produced by these bacteria. However, these steps are rare events: 1 to 10% of the vector molecules contain an insert at the end of the experiment and 1 to 10% of bacteria receive effectively a DNA molecule (vector alone or vector+insert). Thus, it is necessary to perform an efficient selection step to isolate bacteria containing a vector and if possible, bacteria containing a vector with insert.
Antibiotic resistance is widely used and validated as selection-pressure marker for selection of bacteria containing a vector. Antibiotic resistance gene is added to the vector backbone. Only a bacterial cell having incorporated a plasmid encoding for a resistance to one specific antibiotic will divide and/or survive in a medium containing a sufficient amount of this antibiotic, allowing to easily select transformed host bacterial cells, and to maintain them in culture.
However, the use of antibiotics and antibiotics resistance sequences present several drawbacks.
Firstly, the transfer of this antibiotic(s) resistance to other bacteria is possible. Indeed, bacteria are exchanging DNA even between different species (conjugation, transduction, passive or active DNA uptake). During these DNA exchanges, plasmids play a major role especially in the case of DNA conjugation. When antibiotic(s) resistance genes are used in recombinant DNA techniques, plasmids contain these genes and when used in applications related to humans or to animals (food industry, DNA vaccine, etc.), it exists a risk to obtain a transfer of these resistance genes to other bacteria including pathogen bacteria.
Secondly, the use of antibiotics in recombinant DNA techniques is not always very efficient (due to spontaneous resistant mutants for example) and could be costly. Moreover, some antibiotic resistance have been suggested to burden a lot of the host cell energy limiting the efficacy of the host cell to produce biopharmaceutical compounds.
More recently, the tendency is to avoid the use antibiotics in applications related to human or animals (from the food industry to the production of vectors, or vectors for DNA vaccination). Consequently, the FDA (Food and Drug Administration in USA) and EMA (European Medicine Agency) recommend avoiding the use of antibiotics in this kind of applications.
Besides antibiotics, several operons are known to encode both a protein that is toxic to a cell and its specific antitoxin.
These systems, sometimes called “poison-antipoison,” “Toxin-Antitoxin,” or “plasmid addiction,” are now used for plasmid stabilization.
The U.S. Pat. No. 5,888,732, which is incorporated by reference in its entirety, discloses the “Gateway technology,” wherein a donor vector comprises a donor insert (e.g. a DNA fragment of interest), placed between two recombination arms that do not recombine with each other. This gene of interest can be inserted at a defined position into a receiving (acceptor) vector having the corresponding recombination arms. In this technology, the donor and acceptor vectors comprise each a different gene encoding a resistance to an antibiotic. When these vectors are transformed in a sensitive host cell, the cells containing one of these vectors are selected by plating it on a medium containing the corresponding antibiotic.
According to one embodiment of the Gateway technology, besides the presence of a selectable marker, the acceptor vector comprises a gene encoding a toxin placed between the recombination arms. After in vitro recombination between the donor and the acceptor vectors, the DNA mixture is transformed into bacterial cells. This DNA mixture contains different kinds of DNA molecules: donor vector, acceptor vector, co-integrate vector (donor vector associated to acceptor vector), acceptor vector with the DNA fragment of interest and by-product (donor vector without the DNA fragment of interest). Thus, it is necessary to select bacteria containing acceptor vector having integrated the DNA fragment of interest during recombination (replacement of the gene encoding the toxin by the DNA fragment of interest). On one hand, plating the transformed bacteria on a medium containing the antibiotic corresponding to the antibiotic resistance gene of the acceptor vector allows to get rid of bacteria having received a donor vector or a by-product. On the other hand, the presence of the gene encoding the toxin in the original acceptor vector eliminates bacteria containing this acceptor vector (without DNA fragment of interest) or co-integrates. Therefore, thanks to the combined use of antibiotics and gene encoding the toxin, only bacteria containing the acceptor vector having integrated the DNA fragment of interest will grow on the plates.
However, none of the prior art discloses efficient and rapid “antibiotic-free” method and tools allowing easy transfer of a DNA fragment of interest from one vector to another by recombination and subsequent selection of the cells having incorporated the desired plasmid (which has incorporated a gene of interest at a correct position and in a correct reading orientation).
Aims of the Invention
The present invention aims to provide new and improved method and tools that do not present the drawbacks of the state of the art and which improve positive selection and consecutive stabilization of recombinant clones, especially a method and tools that are more simple, more efficient, less labor intensive and that present an improved speed and yield and that facilitate nucleic acids cloning and subsequent expression by cells.
More particularly, the aim of the invention is to propose a method and tools that are based upon an “antibiotic-free” efficient selection of recombinant cells having incorporated correctly an (exogenous) insert (possibly encoding a desired molecule of interest) in a vector after recombination, this kind of insert molecule being possibly used as a DNA vaccine or for gene therapy.